Generation of iPSC line from MYH7 R403L mutation carrier with HCM and isogenic CRISPR/Cas9 corrected control

Introduction Hypertrophic cardiomyopathy (HCM) is a relatively frequent (1/500) heart disease that could result in arrhythmia and sudden death. It is mainly caused by sarcomere gene mutations. Disease mechanisms are still poorly known due to limited access to humanized experimental models. Cardiomyocytes derived from iPSc of mutated patients could serve as a platform for modeling the disease and help identifying novel therapeutic strategies. Objective To derive and differentiate into cardiomyocytes an iPSc line from a patient with an HCM hot-spot mutation and its isogenic control. Method From patient's fibroblasts we derived an iPSC line harboring the heterozygous MYH7 R403L (c.1208G > T) mutation (Okita et al). The mutation was corrected using CRISPR/Cas9 editing to produce an isogenic pair. Genomic editing, pluripotency and cardiogenicity of the clones were evaluated. Results The 2 derived iPSc clones proved to be pluripotent after phosphatase alkaline labelling, RT-qPCR on specific mRNA and proteins immunolabelling (Oct4, Nanog, Sox2, SSEA4). High TaqMan hPSC Scorecard confirmed tri-lineage differentiation potential. Normal karyotype indicated genomic integrity and STR analysis of donor fibroblasts and iPSc lines proved filiation of the clones. The mutated clone was electroporated with spCas9 and a gRNA targeting a PAM 3-bp away from the mutated allele and a correcting ssODN DNA donor. Homologous recombination events were selected by allele-specific ddPCR and reversion of the mutation was confirmed by Sanger sequencing. The 2 lines were able to differentiate into beating cellular sheets and cells stained positive for aACTN2 indicating cardiomycocyte differentiation. FACS on cTNT positive cells counts 75% of cardiomyocytes in independent differentiation experiment after 3 weeks. Conclusion We established an isogenic pair of iPSc lines from a HCM patient with MYH7 mutation defining a model for cellular and molecular study of HCM.